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  • Björkstén, Sofie (2011)
    Angiogenesis, the formation of new blood vessels from preexisting vascular network, is an essential process during tumor development. Growing tumors secrete different growth factors that induce angiogenesis, of which vascular endothelial growth factor (VEGF) is predominant. Angiogenesis inhibitors act either by blocking the extracellular bindning of growth factor to its receptor by monoclonal antibodies or by blocking the intracellular signalling pathway by small-molecule agents. The small-molecule agent sunitinib is a multitargeted tyrosine kinase inhibitor that has antiangiogenic and antitumor activities due to the selective inhibition of several tyrosine kinase receptors. Sunitinib is approved for treatment of gastrointestinal stromal tumors, renal cell carcinoma and pancreatic neuroendocrine tumors. Known side effects are hypertension, cardiotoxicity and renal damage. These toxic effects are due to sunitinibs "off-target" toxicity, which occurs when a tyrosine kinase inhibitor causes adverse effects via inhibiton of a kinase not intended to be a target of the drug. For example inhibition by sunitinib of AMPK, a kinase that plays key roles in maintaining metabolic homeostasis in the heart, accounts in part for the toxicity seen in cardiomyocytes exposed to sunitinib. By achieving a better understandning of what causes the side effects it could be possible to develop treatments that reduce off-target effects. Caloric restriction is one nonpharmacological approach that has been shown to have beneficial effects on the heart partly by activating sirtuins. Sirtuins regulate a diverse array of cellular functions, including metabolism, gene transcription, cell division and cellular stress response. The aim for this study was to investigate whether caloric restriction improves sunitinib-induced cardiovascular toxicity and renal damage in rats, and to study activated cellular pathways. In this study 40 spontaneously hypertensive rats (SHR) and 10 normotensive Wistar-Kyoto (WKY) rats were used. They were divided into groups depending on treatment; I WKY control, II SHR control, III SHR + caloric restriction 70 %, IV SHR + sunitinib 3 mg/kg and V SHR sunitinib 3 mg/kg + caloric restriction 70 %. The follow-up period was eigth weeks. Blood pressure was messured weekly, metabolic cages were used week 4 and week 8 for urine samples, echocardiography was performed the last week and vascular response was studied at the end. The proteins Sirt1 and AMPK in heart were investigated by Western blot and the amount of the marker of macrofage ED1 in kidney by immunohistochemistry. Based on this study it was observed that the dose 3 mg/kg sunitinib was well tolerated in rats because it did not cause more extensive hypertension, worse hypertrophy or renal damage compared to untreated SHR groups. This study also showed that short-term caloric restriction has beneficial cardiovascular effects.
  • Salminen, Sanna (2011)
    The background of this study is increase in the ageing population and in medication use. Aged-related changes in pharmacodynamics and pharmacokinetics may change medication response in elderly patients and lead to adverse reactions. For elderly people the risk of being hospitalized due to adverse drug reactions is four times higher than for younger people. Many of these problems could be prevented by avoiding the use of certain drugs in the elderly. Several criteria have been developed to assess medication appropriateness in the elderly. The aim of this study was to develop a new Finnish Medication Risk Assessment (MRA) tool to be used by trained nurses to assess the presence of risks related to use of medicines in outpatients aged 65 years and older. A preliminary tool was developed through a comprehensive literature review of tools to indicate appropriateness and risks of elderly medications, and through expert opinions. The tool was then validated by using three-round Delphi-method. Delphi-method is a qualitative consensus method which is based on group judgement of a subject matter. The first and the second Delphi-rounds measured the tool's suitability and the third Delphi-round measured the importance of the items of the tool in estimating risks related to the use of medications of elderly patients. In this study, 33 expert geriatric panelists were approached of whom 11 physicians, three pharmacists MSc (Pharm.) and four nurses agreed to participate. The results from the Delphi-rounds were evaluated both quantitatively and qualitatively. Through the three-round Delphi-method was developed a MRA -tool that contains 19 items. According to the panelists the items of the tool are either important or moderately important. This indicates that the tool is valid to estimating medication risks in use of medications in this population. Further studies are needed to test the tool among nurses and patients. The MRA -tool was primary developed for estimating risks in medication use, but it could also be used for educational purposes. In the future, it is possible to implement safer and more appropriate pharmaceutical treatment for elderly patients by using this Medication Risk Assessment -tool.
  • Niskanen, Anna (2013)
    Polypharmacy and age-related changes in pharmacodynamics and pharmacocinetics may lead to drug-related problems in elderly patients. Accurate medication reconciliation and medication review on admission may help to control drug-related problems and optimize drug therapy in elderly patients. Several models have been developed to reconciliate and review medications at this point of care. A Finnish model can be developed on the basis of the se models. The aim of this study was to develop a tool for medication reconciliation and medication review on admission for ward pharmacists’ use in the Lahti city hospital. The tool was developed with an action research method in cooperation with the multiprofessional study group. A preliminary tool was developed through doctors’ (n = 2), nurses’ (n = 3) and ward pharmacists’ (n = 2) interviews, a literature review and the expertise of the multiprofessional study group. The preliminary tool was piloted twice in the Lahti city hospital. After the first pilot a view changes were made to the too l by the experiences of the ward pharmacists. Doctors (n = 3) who worked at the study ward during the first pilot were interviewed to find out their views on the medication reconciliation and medication review process so that their views could be taken into consideration in the development of the final version of the tool. After second pilot ward Pharmacists (n = 2), researchers (n = 2) and an expert of geriatrics from the study group took part in a group conversation. Through the group conversation and doctors’ interviews was developed the final version of the tool. The developed tool contains sections for patient’s background information, patient interview, medication reconciliation, drug-related problems, proposed medication changes and doctor’s decisions on the proposed changes. Also instructions of the medication reconciliation and medication review process were developed for ward pharmacists. The developed tool will be used in an intervention study in the Lahti city hospital. In the future a new version of the tool could also be developed to be used in other hospitals in Finland to reconciliate and review medications at the time of hospital admission.
  • Kleme, Jenni (2012)
    The medicines information and counseling given by health care professionals are essential in supporting patients' medication therapy. Given that medication therapies are often associated with medicine-related problems among the elderly, proper knowledge on medicines and their use is especially important for this particular patient group. Benzodiazepines and related drugs are of special concern in the elderly. Despite the current care guidelines, they are commonly used by the elderly, often also regularly and long-term basis. Benzodiazepines and related drugs are associated with multiple potential adverse drug reactions that their elderly users should be aware of. This study aimed at assessing the knowledge on medications, and needs and sources of medicines information on benzodiazepines and related drugs in the elderly. Especially, medicines information related to benefits and adverse drug reactions was studied. Additionally, data on use and subjective experiences of benzodiazepines or related drugs in the elderly were explored. Structured interviews were conducted among patients aged 65 years and using benzodiazepines or related drugs (n = 38) in acute wards (n = 2) of Pori City Hospital in 2004. Elderly patients reported that the package leaflet was the main source of medicines information on benefits and adverse drug reactions relating to medicines they used. The physician was reported as a second source after the package leaflet. More than 50 percent of the elderly (n = 20) had not received information about the benefits or adverse drug reactions of benzodiazepines from their health care providers or relatives. The information received had merely focused on benefits of drug than adverse drug reactions. Most commonly the elderly (61 %, n = 23) knew, that the use of benzodiazepines can cause drug dependence. Least commonly, they were aware that benzodiazepines can cause muscular weakness, depression and falling over. Eight elderly were not aware of any asked adverse drug reactions and nearly two thirds of the patients (63 %, n = 24) knew less than four adverse drug reactions out of eleven. The results indicate that elderly patients are not well aware of the effects of benzodiazepines and related drugs they use. Additionally, they may more often receive information from the package leaflet than health care professionals. Physicians and other health care professionals should pay more attention to counseling elderly patients especially about the benefits and adverse drug reactions of benzodiazepines and related drugs.
  • Wikman, Essi (2019)
    Streptococcus pneumoniae is a bacterium that causes invasive pneumococcal disease (IPD) such as bacteraemia and meningitis, and pneumonia. The prevalence of pneumococcal diseases is high in infants and in ≥65-year-olds. Also, the incidence of pneumococcal disease is higher in medical risk groups compared to the base population. Pneumococcal diseases can be prevented by vaccinations and since 2010 pneumococcal vaccine PCV10 has been in the national vaccination programme for infants in Finland. The aim for this study is to evaluate the cost-effectiveness of pneumococcal vaccinations in national vaccination programme for the 65-year-olds in medical risk groups (diabetes, chronic coronary artery disease, asthma and COPD). Secondary aim is to examine uncertainty factors that are related to economic evaluations of pneumococcal vaccinations in the elderly. Cost-utility analysis was used as the economic evaluation method. It is a method where health gains are measured by quality-adjusted life years (QALYs). Static multicohort model was chosen for the modelling. Some of the used parameters were acquired from the literature and most of the epidemiology and cost parameters were acquired from research reports and articles published by National Institute for Health and Welfare. Analyses were made for both pneumococcal vaccines that are registered for adults (PCV13 and PPV23) and in 2 different scenarios: Finland’s present situation where PCV10 is in the vaccination programme for infants (scenario A), and hypothetical situation where PCV13 would be in the vaccination programme for infants (scenario B). Based on the analysis, when PCV10 was in the vaccination programme for infants (scenario A), vaccinating 65-year-olds in medical risk groups was cost saving intervention in the health care perspective for both vaccines in chronic coronary artery disease and asthma and COPD risk groups. In diabetes risk group the costs per QALY’s gained were 2 100 € in scenario A. When PCV13 was in the vaccination programme for infants (scenario B), costs per QALY’s gained for PCV13 vaccinations were: diabetes 52 400 €, chronic coronary artery disease 35 900 € and asthma and COPD 22 000 €. The uncertainty of results was tested with deterministic and probabilistic sensitive analysis. In scenario B the results were sensitive for the waning of the PCV13 produced immune protection, the price of the vaccine, the proportion of pneumonia caused by S. pneumoniae, the changes in the pneumococcal disease incidences and the effect that pneumonia has for the health related quality of life. The cost-effectiveness of vaccinating 65-year-olds with pneumococcal vaccines was different depending on the risk group and on which pneumococcal vaccine is in the vaccination programme for infants. In addition, there are several uncertainty factors that have an impact on the results of economic evaluation of pneumococcal vaccinations.
  • Vartiainen, Mira (2024)
    Current therapies for depression have limitations in efficacy and delayed onset of action. Rapid-acting antidepressants like ketamine, an N-methyl-D-aspartate receptor (NMDA-R) antagonist, have gathered attention as an improved treatment option. However, the neurobiological mechanism underlying their antidepressant effect remains uncertain. Integral mechanisms of action seem to be alterations in synaptic plasticity, global cortical excitation, and repair of neuronal dysfunctions prevalent in the pathophysiology of depression. Emerging evidence does suggest that antidepressant drugs act by facilitating brain derived neurotrophic factor (BDNF) mediated tropomyosin receptor kinase B (TrkB) signaling. Interestingly, rapid-acting antidepressants seem to increase TrkB-associated signaling after their acute pharmacological effect has dissipated, and when animals become sedated and show various physiological changes associated with deep sleep (e.g., slow wave EEG activity, SWA). Indeed, recently a close relationship between sedation and molecular signaling implicated in antidepressant effects has been discovered. The aim of this study was to explore the relationship between sedation and molecular signaling associated with antidepressant effect. This was carried out by assessing the localization of TrkB-associated phosphorylation signaling in the adult male mice medial prefrontal cortex (mPFC) using dexmedetomidine, a sedative. Key signaling molecules such as ribosomal protein S6 kinase (p70S6K), ribosomal protein S6 (rpS6), glycogen synthase kinase 3 (GSK3), mitogen activated protein kinases (MAPKs) and immediate early gene c-Fos, were examined through immunohistochemical (IHC) analysis. Two separate experiments were conducted using naïve adult 8-13-week-old (n=8 and n=10) male C57BL/6JRccHs mice. In the experiments mice were injected intraperitoneally with either dexmedetomidine (0,05 mg/kg, Dexdomitor®), or saline followed by a 30-minute recovery period whereafter mice were euthanized. In the first experiment, medial prefrontal cortex samples were collected immediately post decapitation for western blot (WB) analysis. The results showed that dexmedetomidine significantly activated TrkB-associated signaling in brain homogenates, consistent with expectations. In the second experiment, mice were perfused with 4% paraformaldehyde (PFA) before brain collection for IHC analysis. However in this experimental setting, no significant difference in the localization of TrkB-associated signaling induced by dexmedetomidine was observed compared to saline. Although, no significant results for signal localization were observed, the results provide insights into the neurobiological effect of sedation induced TrkB-signaling. Further research factoring in limitations is needed to uncover the involvement of physiological states in antidepressant mechanisms.
  • Kivioja, Saara (2023)
    P-glycoprotein (ABCB1, MDR1) is an efflux transporter expressed widely through the body, but mainly focused on tissues that have protective or excretive function, such as liver and blood-brain-barrier. Many clinically used drugs from variety of therapeutic groups are substrates of P-glycoprotein, and changes in the function of P-glycoprotein may have impact on the drugs pharmacokinetics and -dynamics. The impact of genetic polymorphism on P-glycoprotein activity have been investigated for several years, but due to contradictory results no consensus has been made. The aim of this Master’s thesis was to investigate the effect of five different P-glycoprotein single nucleotide polymorphisms (SNPs) on transport activity. The study was performed by Spodoptera frugiperda (Sf9) membrane vesicles expressing P-glycoprotein variants. Baculovirus-derived expression system was used to introduce the ABCB1 gene to the cells. Vesicle assay was performed with N-methylquinidine (NMQ), and ATP-dependent transport of P-glycoprotein variants was compared to the reference gene. Amino acid change Cys717Tyr led to no transport activity compared to reference gene, and Arg669Cys associated with higher transport activity of NMQ. Arg588Cys, Ser795Cys and Ile836Val indicated no effect on the transport activity. Other aim for this Master’s thesis was to create a new in-house protocol to study P-glycoprotein polymorphism in vitro. Substrate accumulation assay for Rhodamine-123 in Sf9 cells analysed with flow cytometry was established, as flow cytometry is widely used method in other laboratories to study P-glycoprotein polymorphism. The baseline for flow cytometry assay was created successfully by optimizing substrate concentration and incubation time. According to the results, SNPs can impair P-glycoprotein function. New method to study P-glycoprotein function was created, and this method can be used to further study the effects of genetic polymorphism of P-glycoprotein and to compare the result between studies. The results gained from these in vitro studies can be utilized to understand in vivo pharmacogenetic findings.
  • Tikkanen, Alli (2019)
    Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is an influx transporter expressed widely throughout the body in tissues such as intestine, liver, brain, placenta and skeletal muscle. Since many clinically used drugs are transported by OATP2B1, changes in the function of the transporter due to genetic polymorphism could lead to altered pharmacokinetics or -dynamics of OATP2B1 substrate drugs. The aim of this Master’s thesis was to create and optimize a cellular uptake assay to study the function of OATP2B1. Furthermore, the aim was to study the effects of six naturally occurring nonsynonymous single nucleotide variants on OATP2B1 transport function in vitro. With site-directed mutagenesis, single nucleotide changes were introduced into the gene coding for OATP2B1. OATP2B1 variants were expressed in human derived HEK293 cell line using baculovirus expression system. A cellular uptake assay with estrone-3-sulfate and a fluorescent probe 4’, 5’-dibromofluorescein (DBF) as substrates was set up and optimized. With the assay, OATP2B1-mediated uptake of variants was compared to the transport activity of OATP2B1 wild type. Amino acid changes Ser486Phe and Cys520Ser impaired OATP2B1 transport function severely. In addition, variant Thr318Ile transported DBF and estrone-3-sulfate less efficiently compared to OATP2B1 wild type, but Arg312Gln, Thr392Ile and Ser532Arg transport function was not affected. A method to study OATP2B1 function was created successfully. According to the results, single amino acid changes in OATP2B1 can impair OATP2B1 function. The results and method can be utilized to understand findings from pharmacogenetic studies in vivo, and to predict consequences of especially rare variants, which can be difficult to detect in small sample populations in clinical studies. However, further studies on the expression level and cellular localization of OATP2B1 variants are needed to fully characterize the impact of the variants studied.
  • Somersalo, Petter (2017)
    Cells release different types of phospholipid bilayer-limited vesicles into the extracellular space. These are commonly referred to as extracellular vesicles (EVs). Exosomes (EXOs), ca 50-100 nm in diameter and microvesicles (MVs), ca 100-1000 nm in diameter, having different intracellular origin, are the two main subpopulations of EVs. EVs have been demonstrated to carry a range of proteins and nucleic acids subsequently delivered to recipient cells, making them attractive as drug delivery vehicles. Several mechanisms for the cellular uptake of EVs have been established. When a nanoparticle is introduced into blood plasma, plasma proteins are adsorbed to its surface, forming a protein corona. The formation of the corona is a dynamic process, governed by individual protein concentrations as well as their respective affinities for the surface. Proteins of the corona interact with surrounding cells, thus being able to influence the cellular uptake of the nanoparticle. In the current study, the uptake of PC-3-derived EVs into PC-3 cells was investigated. Moreover, the impact of a human blood plasma-derived protein corona on said uptake was assessed. EVs were isolated from collected PC-3 cell culture medium using differential centrifugation. Experiments were performed separately for MVs (20000xg EV-fraction) and EXOs (110000xg EVfraction). SDS-PAGE analysis revealed adsorption of plasma proteins to EVs, following their exposure to plasma. Prior to uptake experiments DiO-labelled EVs were either incubated or not incubated in plasma. Plasma incubation lasted overnight. PC-3 cells were then treated with either of the two EV-preparations. Following incubation, EV uptake was assessed using confocal microscopy by determining the percentage of positive fluorescent cells in cell cultures. Pre-study plasma incubation resulted in a reduced or unchanged uptake of MVs and in a reduced uptake of EXOs, when compared to their native counterparts. In conclusion, the plasma-derived protein corona was shown not to improve EV uptake. It is worth noting that the current study limits itself to the use of PC-3-derived EVs and PC-3 cells as recipient cells in uptake experiments.
  • Hietala, Tarja (2017)
    Twin screw granulation (TSG) has gained considerable interest as a continuous wet granulation method in the pharmaceutical industry and has been studied the most. However, there is still lack of understanding how continuous granulation affects the material compaction behavior even though it has been noticed in several dry and batch wet granulation studies that the granulation process has an influence on the final tablet strength. Thus, studies on the material compactability and tabletability after continuous wet granulation are relevant for the overall understanding of twin screw granulation process and its effect on material behavior in tableting. Hence, the main objective of this study was to investigate the influence of continuous twin screw granulation on the compactability and tabletability of commonly used excipients. Additionally, the impact of binder on the compaction behavior of materials was examined. Furthermore, the suitability of two "loss in compressibility" models i.e. the Unified Compaction Curve (UCC) model and a porosity model to predict the loss in tablet strength after twin screw granulation and for the materials used was assessed. Earlier, the models have been applied to dry and batch wet granulations only. Full factorial design of three variables (binder type, binder addition method and the number of kneading elements) with two levels was conducted for the ConsiGma1 twin screw granulation of formulations containing microcrystalline cellulose (MCC), mannitol or anhydrous dicalcium phosphate (DCPA) as the main excipient and polyvinylpyrrolidone (PVP) or hydroxypropyl cellulose (HPC) as binder. Magnesium stearate was added as lubricant after granulation prior to tableting. In addition to the full factorial design, granulation with PVP, dry binder addition and four kneading elements was repeated for each main excipient. In total this made 27 experiments. The granules were dried and milled after granulation and all the batches were tableted. Additionally, all the formulations were direct compressed in order to be able to detect the change in compactability and tabletability after granulation. Torque of the granulation was determined as well as bulk density and particle size distribution of the granules. Additionally, the tensile strength and porosity of the tablets were analysed. Tabletability and compactability were determined based on the compaction pressure and the obtained tensile strength and porosity values of the tablets. Furthermore, parameters (PWG, TWG and εWG) describing the loss in compressibility models were calculated. MCC experienced loss in compactability and tabletability after twin screw granulation due to hornification effect. On the other hand, the compaction behavior of mannitol improved due to the formation of porous granules. The compactability of DCPA decreased and the tabletability increased. However, the change was only moderate presumably due to brittle nature of DCPA. Additionally, the binder type had an effect of the compaction behavior of the materials, PVP producing stronger tablets compared with the less hydrophilic HPC. However, the binder addition method played only a small role in modifying the compaction behavior. The UCC model was applicable to MCC as loss in tabletability was detected. Thus, the model can be used to predict tablet tensile strength when MCC is granulated with twin screw granulator. Additionally, the UCC model can be used to design the granulation process to achieve a target tensile strength based on small scale preliminary studies thus reducing the resources needed for case-studies. However, the UCC model was not feasible to mannitol and DCPA because they experienced improvement in tabletability after twin screw granulation. The porosity model was applicable to MCC and DCPA but not to mannitol as it showed improvement in compactability. The porosity model described the loss in compactability of MCC only moderately due to lack of tensile strength data points and the linearity of the tensile strength-porosity relationship. However, the model described well the loss in compactability of DCPA at tablet porosities achieved with compaction pressures used in industry. As a conclusion, the results demonstrate that twin screw granulation can have a significant impact on the final tablet strength and that the compaction behavior of the formulation can change either way depending on the used materials. Furthermore, the small influence of the binder addition method on the tablet strength indicates that the time consuming binder dissolving process step can be excluded from the tablet production chain enabling continuous manufacturing with twin screw granulation.
  • Noponen, Henna (2024)
    Indomethacin is a poorly soluble but highly permeable drug, and its biological availability can be improved by enhancing its solubility. In this study, co-crystals and co-amorphous systems of indomethacin and nicotinamide were prepared in a 1:1 molar ratio, which have previously been shown to enhance the solubility of indomethacin. It has also been observed that the co-amorphous indomethacin-nicotinamide system crystallizes into a co-crystal during storage. This study aimed to further investigate the properties, solubility, and stability of these compounds, and tablet formulations were also prepared from the co-crystal and co-amorphous systems. Powdered co-crystals and co-amorphous systems, as well as tablets prepared from them, were stored at 25°C with 60% relative humidity and at 40°C with 75% relative humidity, and their solubilities were studied for 12 weeks. The stability of the samples was also examined using Fourier infrared spectroscopy and differential scanning calorimetry over the same period, and changes in the physical properties of the tablets were monitored throughout the study period. Additionally, the effect of HPMC on the prevention of indomethacin recrystallization was investigated. Both the co-amorphous and co-crystalline forms were found to enhance the solubility of indomethacin in both powder and tablet formulations in this study. The co-crystal was stable, with no changes observed in its crystal structure or solubility over the 12-week study period. However, handling the co-amorphous material turned out to be difficult due to its low glass transition temperature of 19.68°C, causing the powder to soften at room temperature. During storage, it was shown to crystallize into a co-crystal, but its solubility properties were weaker to those of the actual co-crystal. None of the solubility tests showed evidence of indomethacin recrystallization, so the potential effect of HPMC on this phenomenon could not be determined in the study. Warmer and more humid conditions were found to increase the tensile strength of the tablets, resulting in slower dissolution.
  • Rossi, Vilma (2020)
    Background: Inhaled therapy is the most widely used treatment for asthma and chronic obstructive pulmonary disease (COPD). Inhaled medicinal product has several advantages, including high local drug concentration in the lungs and reduced systemic adverse effects. However, the challenge with inhaled therapy is that many asthma and COPD patients do not know how to use their inhaler properly. Suboptimal inhaler use can lead to poor clinical control. The Association of Finnish Pharmacies has developed inhalation technique assessment service (ITAS) to detect and correct patients’ inhalation technique and to give information regarding the inhaler and inhaled therapy, such as drug storage and oral care. Objective: The aim of the study is to investigate whether asthma and COPD patients’ ability to prepare the Respimat inhaler and the patients’ ability to properly inhale the drug improve after receiving ITAS. The second objective is to find out what patients and pharmacists think about the service and which customer groups benefit the most from the service. Methods: The study design is an uncontrolled pre-post intervention. 33 pharmacies participated in the study. All patients who were buying a prescribed Respimat inhaler, were offered to participate in the study. Patients’ inhalation technique was assessed before (baseline) and immediately after ITAS (follow up 1). In addition, the inhalation technique was assessed the next time the patient came to pharmacy to buy Respimat inhaler (follow-up 2). Questionnaires were used to assess patients’ and pharmacists’ perceptions of ITAS. Results: 228 baseline and follow-up ITAS were performed. The results of follow-up 2 will be published later in a separate article. 14 % of the patients performed all the steps (both inhaler preparation before first inhalation and inhalation process itself) correctly at baseline. After ITAS the number increased to 77 %. At baseline 30 % of the patients had an optimal inhalation technique (all inhalation steps correct) and after ITAS the number increased to 85 %. 70 % of the patients had an acceptable technique (all critical steps correct) before and 93 % after ITAS. Both patients and pharmacists felt that the service was beneficial to the patients when thinking the proper inhaler preparation and proper inhalation technique. Overall patients’ and pharmacists’ satisfaction were high towards ITAS. Our study indicates that patients benefit from ITAS regardless of patient’s age or how long the patient have been using the Respimat inhaler. Conclusions: A pharmacist-led inhalation technique assessment service significantly improves asthma and COPD patients’ inhalation technique with Respimat inhaler. ITAS should be performed regularly as part of the delivery of the inhaled drug to the patient. Further research is needed on the effectiveness of ITAS with other inhalers.
  • Vähä-Mäkilä, Maria (2012)
    The aim of this master`s thesis was to investigate the accuracy of in silico inhalation model to predict pharmacokinetics of orally inhaled products. In literature review special features of the inhalation medication and current statements of medicinal regulatory agencies about bioequivalence (BE) of inhaled products are discussed. The ability of generalized pharmacokinetic BE studies to replace the traditional efficacy studies is a major question in the regulatory agencies. Also the usefulness of published in vitro - in vivo correlations (IVIVC) as an aid for inhaled product development in pharmaceutical industry is considered. Furthermore the most commonly used in silico lung deposition models and their properties are presented. In the experimental part a generic in silico inhalation model was constructed using a proper software and Orion Oyj`s in vitro and in vivo research materials on certain dry powder inhaler (DPI) products. Based on in vitro knowledge the aim of modeling was to predict the pharmacokinetic behavior of a therapeutic drug used in inhaled products. Also the applicability as a tool in clinical study design of inhaled products was estimated. Inhalation model consisted of two separate modeling parts utilizing primary in vitro characterization results of DPI products. Lung deposition of products was predicted with the ARLA (The Aerosol Research Laboratory of Alberta) respiratory deposition calculator available to the public while drugspecific pharmacokinetics was simulated using constructed Stella model (isee systems). ARLA lung deposition model takes into account several factors affecting the final lung dose of medical aerosol. Those include aerosol formulation and the dimensions of the device, as well as breathing conditions and inhalation mode. A rough sensitivity analysis was carried out with ARLA considering the effect of these factors on predicted lung deposition fractions. The predicted plasma concentration profiles, Cmax and AUCt values of the model drug were markedly lower than the experimental values. ARLA deposition model predicted moderately the order of systemic drug exposure obtained with different DPI products. The inhalation model built in the experimental part needs to be refined using more comprehensive and trustable source and reference material. The role of clinical BE studies in the marketing approval of generic inhalation product will be important because currently in silico predictions are still under development.
  • Myllymäki, Pilvi (2018)
    The majority of potential new chemical entities reaching drug development phase belong to Class II the Biopharmaceutics Classification System (BCS) which complicates formulation of orally administered drugs. Therefore, there is a need to develop methods to increase the solubility and dissolution rate. Transformation of a crystalline drug into its amorphous form can be used to enhance these properties of poorly water-soluble drugs. However, amorphous drugs are thermodynamically unstable and tend to recrystallize back to the crystalline form. Coamorphous forms are a new and promising method to stabilize amorphous form. A relatively new approach is to combine the active drug compound with an amino acid to form a coamorphous system. In this study, co-amorphous systems were prepared from gamma, alpha or amorphous form of indomethacin (IND) and tryptophan (TRP) by ball milling. The solid-state changes during milling were investigated to obtain information about the co-amorphization process. The main objective was to investigate the effects of initial solid state of indomethacin on the formation pathways. In addition, different analytical methods were compared with respect to observed endpoints of the formation process. Raman spectroscopy has not been used in previous studies regarding solid state changes in co-amorphous forms. The presence of fluorescence in amorphous systems may have limited use of the method. A time-gated Raman setup together with X-ray powder diffraction and differential scanning calorimetry (DSC) was used to investigate this kind of potentially fluorescent system. Principal component analysis of spectral data revealed that the three different binary systems had individual and direct pathways towards the same end points during milling. This indicates that the co-amorphous form formed after 60 minutes of ball milling is not dependent on the initial solid-state form of IND. Straight pathways also indicated direct transformation to the coamorphous form. DSC was found to be the most sensitive method to detect changes for the longest period during co-amorphization. Conventional Raman spectroscopy was found to be suitable for investigation of the co-amorphization process. However, time-gated Raman spectroscopy did not show significant advantages compared to conventional Raman data. This study revealed that the most stable form of IND could be used for production of co-amorphous form together with TRP. Raman spectroscopy could potentially be used for investigating coamorphization also as an in-process analytical method.
  • Lindstedt, Hanna (2022)
    Drug-induced liver injury (DILI) is a relatively rare hepatic condition that can be classified as predictable and unpredictable. However, DILI is a primary reason for drug withdrawals, post-marketing warnings, and restrictions of use. DILI is a problem for the drug users but also for the pharmaceutical industry and regulatory bodies. From the perspective of patients' and clinicians', DILI is the major cause of acute liver injury. At present, a major problem predicting DILI in drug discovery is a poor understanding of its mechanisms as well as the complexity of DILI pathogenicity. The main mechanism behind DILI are alterations in bile acid homeostasis, oxidative stress, and mitochondrial dysfunction. More than 50 % of drugs causing DILI are causing mitochondrial impairment. If the normal function of mitochondria is disturbed, the energy production of the cell decreases, and cell function decline leading eventually to the cell death. In this study prediction of mitochondrial toxicity was studied using cryopreserved primary hepatocytes of humans and rats. The aim of the study was to clarify if there are interspecies differences in the prediction of toxicity but also investigate possible differences in the mechanisms behind hepatotoxicity by using three well-known compounds toxic to mitochondria. To determine these differences, total cellular ATP was measured after 2- and 24- hour exposure time to gain information on overall viability and possible adaptive responses. Mitochondrial energy pathways were studied as a real-time monitoring acute exposure of test compounds. Morphology, location, and possible adaptive response of mitochondria were studied using a fluorescent probe and antibody staining combined with high content imaging (HCI). Overall, primary rat hepatocytes were more sensitive to the test compounds than human hepatocytes. Also, there were differences between human hepatocyte batches that may reflect the metabolic differences between hepatocyte donors. Immunolabeling did not bring any additional values compared to the fluorescent probe staining in the study of morphology of mitochondria. Additionally, it was noticed that treatment with paraformaldehyde significantly changed the hepatocyte mitochondria morphology. Overall, more effort is needed to develop image analysis of mitochondria morphology. Finally, studying mitochondrial morphology has proven to be difficult, and this study did not unfortunately reveal any information about the adaptive responses of mitochondria for drug-induced liver injury.
  • Huusko, Karoliina (2013)
    Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.
  • Mustonen, Merja (2011)
    Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.
  • Sjöstedt, Noora (2011)
    The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.
  • von Schantz, Sofia (2015)
    This study aims to address how easily an individual with no prior inhaler experience can learn to use a dry powder inhaler (DPI) through video education. This is a comparative study of four DPIs (Diskus, Easyhaler, Ellipta and Turbuhaler). Different properties affecting ease of use, patient preference as well as educational videos as a method of providing inhaler instructions were investigated. The study used a triangular methodology. The sample consisted of 31 individuals (24-35 years). All participants were considered inhaler naïve. After watching the video education material for a particular inhaler the participants' demonstrated the use of it. Educational videos for all four inhalers were watched and use of all placebo inhalers was demonstrated in a random order. These demonstrations were videotaped. The demonstrations were thereafter checked against a predefined checklist and all mistakes were recorded. Only 33 % of inhaler demonstrations were completed without the participants making any mistakes that could compromise the efficacy of the inhaled medication in a real-life situation. The frequency of error varied greatly between different types of inhalers. Ellipta proved to be most often used correctly with 55 % demonstrating use without making any mistakes. This was closely followed by Diskus for which 48 % demonstrated correct use. The difference between the average error frequency for Ellipta and Diskus was statistically insignificant. With Easyhaler 19 % percent of participants were able to demonstrate correct use, the corresponding percentage for Turbuhaler was 16 %. When comparing participants' demonstrations for Easyhaler and Turbuhaler, the difference in average error frequency between the devices were not statistically significant. The average frequency of error was lower when using Ellipta in comparison to Easyhaler and Turbuhaler (statistically significant). The same indications were found when comparing average frequency of error for Diskus, to those for Easyhaler and Turbuhaler. Comparing the participants self-reported correct use against the actual numbers it is clear that participants often thought they were using the inhaler correctly when they in fact were not. When asked to rank the inhalers from most preferred to least preferred, Ellipta emerged as a favorite. Turbuhaler received the second highest scores, Diskus the third and Easyhaler was least preferred. However, only the difference between preference scores for Ellipta and Easyhaler was deemed statistically significant. The high frequency of error suggests that even though participants generally considered the inhalers intuitive and easy to use, they would have required more comprehensive inhaler education in order to achieve correct inhaler technique. Further, the results indicate that video demonstrations are not ideal for providing inhaler education for first time inhalers users. The most prominent problem with video education is that it provides no feedback to the user regarding their inhaler technique. This may present real problems as the results of this study show that participants tended to overestimate their own inhaler technique. Patient education plays a central role in asthma care and needs to be given proper attention even though the inhalers might be considered intuitive and easy to operate. Interesting areas for future research include investigating interactive learning videos as a way of improving video education on inhaler technique.